The present invention relates to transgenic animal models for cardiac hypertrophy. It further relates to the use of such transgenic animals to detect substances with therapeutic activity toward cardiac hypertrophy. It further relates to assays to detect substances for use in treating cardiac hypertrophy. It further relates to methods for treating cardiac hypertrophy.
Cardiac Hypertrophy
Cardiac hypertrophy is an adaptive response of the heart to virtually all forms of cardiac disease, including those arising from hypertension, mechanical load, myocardial infarction, cardiac arrhythmias, endocrine disorders, and genetic mutations in cardiac contractile protein genes. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to dilated cardiomyopathy, heart failure, and sudden death. In the United States, approximately half a million individuals are diagnosed with heart failure each year, with a mortality rate approaching 50%.
Despite the diverse stimuli that lead to cardiac hypertrophy, there is a prototypical final molecular response of cardiomyocytes to hypertrophic signals that involves an increase in cell size and protein synthesis, enhanced sarcomeric organization, up-regulation of fetal cardiac genes, and induction of immediate-early genes, such as c-fos and c-myc. See, Chien et al. (1993) Ann. Rev. Physiol. 55:77-95; and Sadoshima and Izumo (1997) Ann. Rev. Physiol. 59:551-571. The causes and effects of cardiac hypertrophy have been extensively documented, but the underlying molecular mechanisms that couple hypertrophic signals initiated at the cell membrane to the reprogramming of cardiomyocyte gene expression remain poorly understood. Elucidation of these mechanisms is a central issue in cardiovascular biology and will be critical for designing new strategies for prevention or treatment of cardiac hypertrophy and heart failure.
Numerous studies have implicated intracellular Ca2+ as a signal for cardiac hypertrophy. In response to myocyte stretch or increased loads on working heart preparations, intracellular Ca2+ concentrations increase (Marban et al. (1987) Proc. Natl. Acad. Sci. USA 84:6005-6009; Bustamante et al. (1991) J. Cardiovasc. Pharmacol. 17:S110-S113; and Hongo et al. (1995) Am J. Physiol. 269:C690-C697, consistent with a role of Ca2+ in coordinating physiologic responses with enhanced cardiac output. A variety of humoral factors, including angiotensin II (AngII), phenylephrine (PE), and endothelin-1 (ET-1), which induce the hypertrophic response in cardiomyocytes, also share the ability to elevate intracellular Ca2+ concentrations. Karliner et al. (1 990) Experientia 46:81-84; Sadoshima and Izumo (1993) Circ. Res. 73:424-438; Sadoshima et al. (1993) Cell 75:977-984; and Leite et al. (1994) Am. J. Physiol. 267:H2193-H2203.
Hypertrophic stimuli result in reprogramming of gene expression in the adult myocardiumn, such that genes encoding fetal protein isoforms like xcex2-myosin heavy chain (MHC) and xcex1-skeletal actin are up-regulated, whereas the corresponding adult isoforms, xcex1-MHC and xcex1-cardiac actin, are down-regulated. The natriuretic peptides, atrial natriuretic factor (ANF), and b-type natriuretic peptide (BNP), which decrease blood pressure by vasodilation and natriuresis, are also rapidly up-regulated in the heart in response to hypertrophic signals. Komuro and Yazaki (1993) Ann. Rev. Physiol. 55:55-75. The mechanisms involved in coordinately regulating these cardiac genes during hypertrophy are unknown, although binding sites for several transcription factors, including serum response factor (SRF), TEF-1, AP-1, and Sp1, are important for activation of fetal cardiac genes in response to hypertrophy. Sadoshima and Izumo (1993); Sadoshima et al. (1993); Kariya et al. (1994) J. Biol. Chem. 269:3775-3782; Karns et al. (1995) J. Biol. Chem. 270:410-417; and Kovacic-Milivojevic et al. (1996) Endocrinol. 137:1108-1117. Most recently, the cardiac-restricted zinc finger transciption factor GATA4 has also been shown to be required for transcriptional activation of the genes for Ang II type 1a receptor and xcex2-MHC during hypertrophy. Herzig et al. (1997) Proc. Natl. Acad. Sci. USA 94:7543-7548; Hasegawa et al. (1997) Circulation 96:3943-3953; and Molkentin and Olson (1997) Circulation 96:3833-3835.
A number of intracellular signaling pathways have been implicated in transduction of hypertrophic stimuli. For example, occupancy of the cell surface receptors for AngII, PE, and ET-1 leads to activation of phospholipase C, resulting in the production of diacylglycerol and inositol triphosphate, which in turn results in mobilization of intracellular Ca2+ and activation of protein kinase C (PKC). Sadoshima and Izumo (1 993); Yamazaki et al. (1996) J. Biol. Chem. 271:3221-3228; and Zou et al. (1996) J. Biol. Chem. 271:33592-33597. There is also evidence that the Ras and mitogen-activated protein (MAP) kinase pathways are transducers of hypertrophic signals. Thorburn et al. (1 993) J. Biol. Chem. 268:2244-2249; and Force et al. (1996) Circ. Res. 78:947-953. The extent to which these signaling pathways are coordinated during cardiac hypertrophy is unknown. However, all of these pathways are associated with an increase in intracellular Ca2+, consistent with a central regulatory role of Ca2+ in coordinating the activities of multiple hypertrophic signaling pathways.
In B and T cells, the Ca2+, calmodulin-dependent phosphatase calcineurin has been shown to link intracellular signaling pathways that result in elevation of intracellular Ca2+ with activation of the immune response. Calcineurin regulates immune response genes through dephosphorylation of a family of transcription factors known as NF-ATs (nuclear factors of activated T cells). Rao et al. (1997) Ann. Rev. Immunol. 15:707-747. Once dephosphorylated by calcineurin, NF-AT transcription factors translocate to the nucleus and directly activate immune response genes. Flanagan et al. (1991) Nature 352:803-807; Loh et al. (1996) Mol. Cell. Biol. 16:3945-3954; and Loh et al. (1996) J. Biol. Chem. 271:10884-10891. The immunosuppressant drugs cyclosporin A (CsA) and FK506 suppress the immune response by inhibiting calcineurin""s ability to activate NF-AT transcription factors. Shaw et al. (1995) Proc. Natl. Acad. Sci. USA 92:11205-11209; Loh et al. (1996) J. Biol. Chem. 271:10884-10891.
There are no spontaneous mouse mutations with sufficient similarities to cardiac hypertrophy to be usefil as experimental models. A transgenic rodent line has been produced that overexpresses calmodulin under the control of the human atrial natriuretic factor gene. Gruver et al. (1993) Endocrinology 133:376-388. In these mice, developmental overexpression of CaM in mouse cardiomyocytes produced a markedly exaggerated cardiac growth response, characterized by the presence of cardiomyocyte hypertrophy in regions demonstrated to overexpress CaM and by cardiomyocyte hyperplasia, apparent at early developmental stages. However, the expression of calmodulin in these animals is constitutive and is thus not reflective of physiological conditions in humans. Transgenic mice have been created in which a reporter gene is regulated by a tetracycline-controlled transactivator (tTA), which in turn is under the transcriptional control of 2.9 kb of 5xe2x80x2 flanking sequence from the rat xcex1-myosin heavy chain gene. Yu et al. (1996) Circ. Res. 79:691-697.
Current medical management of cardiac hypertrophy includes the use of three types of drugs: calcium channel blocking agents, xcex2-adrenergic blocking agents, and disopyramide. Kikura and Levy (1995) Int. Anesthesiol. Clin. 33:21-37. Therapeutic agents for heart failure include angiotensin II converting enmine (ACE) inhibitors and diuretics. Other pharmaceutical agents which have been disclosed for treatment of cardiac hypertrophy include angiotensin II receptor antagonists (U.S. Pat. No. 5,604,251); and neuropeptide Y antagonists (International Patent Publication No. WO 98/33791). Despite currently available pharmaceutical compounds, prevention and treatment of cardiac hypertrophy, and subsequent heart failure, continue to present a therapeutic challenge.
Thus, there is a need for the development of new pharmacologic strategies for prophylaxis and treatment of cardiac hypertrophy in humans. In order to develop such strategies, there is a need for animal models which accurately reflect the pathological profile of the disease, to allow identification of novel targets for therapeutic intervention. In addition, there is a need for novel assays that allow identification of potential new therapeutic agents to treat cardiac hypertrophy.
The present invention provides non-human animal models for cardiac hypertrophy. Such models are useful for identifying additional targets for treatment of cardiac hypertrophy, and for testing substances for efficacy in treating cardiac hypertrophy. In one aspect, the invention provides a transgenic non-human animal comprising as a transgene a polynucleotide which, when introduced at an early stage into a fertilized egg or embryo of a mammalian non-human animal, can be functionally integrated into the genome thereof, thereby to produce a transgenic animal, characterized in that the polynucleotide comprises a nucleotide sequence encoding a gene product which modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a cardiac hypertrophic signal, in combination with control sequences, including, for example, a promoter, which direct and regulate expression of the gene in somatic cells of the transgenic animal, particularly in cardiomyocytes.
The present invention encompasses a transgenic animal comprising as a transgene a polynucleotide encoding a gene product that modulates transcription of at least one gene expressed in cardiomyocytes in response to a cardiac hypertrophic signal, wherein the animal has a substantially increased probability of spontaneously developing symptoms of hypertrophic cardiomyopathy or similar heart dysfimction.
The present invention further encompasses a transgenic animal comprising as a transgene a polynucleotide encoding a gene product that modulates transcription of at least one gene expressed in cardiomyocytes in response to a cardiac hypertrophic signal, wherein the animal has a substantially decreased probability of spontaneously developing symptoms of hypertrophic cardiomyopathy or similar heart dysfimction.
Transgenic animals of the invention are useful in identifying novel targets for prophylaxis and therapy of cardiac hypertrophy and cardiac hypertrophy-induced dysfunctions. These animals are also useful for investigative purposes, for examining signal transduction pathways involved in response to hypertrophic signals. These animals are further useful to screen for potential therapeutic agents for treatment and/or prophylaxis of cardiac hypertrophy.
Preferably, the polynucleotide used to generate a transgenic animal of the invention is a recombinant DNA construct in which the promoter and at least some control sequences direct expression of an operably linked coding sequence. In some embodiments, the promoter and control sequences are expressed in a cardiomyocyte-specific manner. The coding sequence is fused to a downstream segment comprising at least a fragment of a eukaryotic gene sequence effective to provide signals for terminating transcription and for controlling processing of transcribed RNA during expression of the encoded gene product. In one embodiment, the promoter is derived from an xcex1-myosin heavy chain gene (xcex1-MHC). In other embodiments, the transcriptional control sequences comprise elements which confer inducible expression on the operably linked coding sequence. In some embodiments, the transgenic animal comprises a transgene which encodes a polypeptide which modulates transcription of at least one cardiac hypertrophy-sensitive gene. In these embodiments, the polypeptide can comprise a wild-type amino acid sequence or a mutant amino acid sequence such that the polypeptide has altered function, such as an altered enzymatic fimction or has altered regulatory properties, including, for example, a dominant negative mutant. In other embodiments, the transgene-encoded gene product is an antisense polynucleotide. In yet other embodiments, the transgene-encoded gene product is a ribozyme.
The constructs are introduced into animal embryos using standard techniques such as microinjection or embryonic stem cells. Cell culture-based models can also be prepared by various methods. For example, cells can be isolated from the transgenic animals or prepared from established cell cultures using the same constructs with standard cell transfection techniques.
The invention further provides a method for producing a transgenic animal, for example a mouse or other rodent, having either a substantially increased probability of spontaneously developing hypertrophic cardiomyopathy or similar heart dysfunction or a substantially decreased probability of spontaneously developing hypertrophic cardiomyopathy or similar heart dysfunction, said method comprising incorporating a polynucleotide as specified above into the genome of a non-human animal such that it is functionally integrated therein. At least in preferred embodiments the nucleic acid is a recombinant DNA which is introduced into a fertilized egg or embryo of the animal in order to become functionally integrated into the genome thereof, said recombinant DNA being characterized in that it contains a segment comprising a gene sequence coding a gene product which of modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal, or for a derivative of such a gene product, which segment is fused to an upstream segment comprising promoter and at least some control sequences effective to direct and regulate expression of said coding, and also, preferably, to a downstream segment comprising a eukaryotic gene sequence or fragment effective to provide signals for termination of transcription and for controlling processing of the transcribed RNA.
Constructs for use in generating transgenic animals can be constructed such that expression of the transgene can be constitutive or inducible. A polynucleotide, for use in generating a transgenic animal, comprises a nucleotide sequence which encodes a gene product which modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. In one aspect, the nucleotide sequence which encodes a gene product which modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal is constitutively expressed. In another aspect, the gene which encodes a gene product which modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal is expressed in a regulatable manner. In another aspect, the gene which encodes a gene product that modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal comprises a portion which encodes a dominant negative mutant of a polypeptide.
In one aspect of the invention, a transgenic animal comprises, as a transgene a gene encoding a gene product that represses the transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. In another aspect of the invention, the transgene-encoded gene product induces the transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. For example, the transcription factor YY1 is a global repressor of cardiac hypertrophic responsive genes, while calcineurin generally induces the transcription of cardiac hypertrophic responsive genes.
Another aspect of the invention relates to cells isolated from the above-described transgenic animals. Preferably, such cells are derived from cardiac tissue and are cardiomyocytes. Cardiomyocytes isolated from the transgenic animals of the present invention can be used to test cardiotherapeutic properties of substances to which the cells are exposed in vitro. Thus, the cells are useful to screen for potential therapeutic agents for treatment and/or prophylaxis of cardiac hypertrophy. Such cells can also be used to identify novel targets for prophylaxis and therapy of cardiac hypertrophy and cardiac hypertrophy-induced dysfunctions. These cells are also useful for investigative purposes, for examining signal transduction pathways involved in response to hypertrophic signals.
The invention further provides methods for screening substances for use in treating a cardiac hypertrophy-induced disease state (dysfunction).
In some embodiments, enzyme-based screening assays are provided, wherein the enzymes used are involved in modulating levels of active product of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. The methods generally involve contacting the enzyme with a substance being tested and measuring an activity of the enzyme, compared to a control sample comprising the enzyme in the absence of the substance being tested.
In other embodiments, cell-based screening assays are provided, wherein substantially isolated cardiomyocytes are contacted with a substance to be tested, followed by measuring a cardiomyocyte function or parameter. An alteration in the cardiomyocyte function being measured, when compared to a control cell sample to which no substance is added, indicates that the substance is suitable for use in treatment of a cardiac hypertrophy-induced dysfunction. In some of these embodiments, the cardiomyocytes are derived from a transgenic animal of the invention. In other embodiments, the cardiomyocytes are derived from a non-transgenic animal.
In still other embodiments, whole animal-based screening assays are provided. In these embodiments, invention encompasses the use of transgenic animals of the invention, or parts thereof, for testing cardiotherapeutic properties of substances administered to said animals, or for testing the activity of such substances in controlling or inhibiting the development of hypertrophic cardiomyopathy. Thus, according to another aspect of the invention, a method of screening and identifying or testing a drug or other substance for activity against the development of or in the treatment of hypertrophic cardiomyopathy, comprising treating a transgenic animal of the invention, or a part thereof, with said drug or other substance concerned and detecting or noting any reduced incidence in the development of hypertrophic cardiomyopathy and reduction in morbidity, as compared with corresponding animals that are not treated with the drug or substance, or detecting or noting an effectiveness in maintaining, restoring or improving heart function.
The invention further provides the use of compositions comprising substances identified by the methods of the present invention, as well as known substances, for treating cardiac hypertrophy and heart failure. Such compositions include substances which increase active levels of one or more gene products which normally repress transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal, thereby reducing the expression of the at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. Such compositions also include substances which reduce active levels of one or more gene products which normally increase transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal, thereby reducing the expression of the at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. Further included are substances which are known modulators of gene products that modulate transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal, for use in treating a cardiac hypertrophy-induced dysfunction. Included are known inhibitors of calcineurin, and derivatives of these inhibitors that inhibit a phosphatase activity of calcineurin.
The invention further provides methods of treating cardiac hypertrophy using modulators of gene products which modulate transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. In one aspect of the invention, calcineurin inhibitors are used to control cardiac hypertrophy and heart failure. The invention further provides methods of treating cardiac hypertrophy, or a cardiac hypertrophy-induced dysfunction, comprising administering to an individual in need thereof a substance which diminishes or reverses the progression of the dysfunction. In some embodiments, the substances inhibit expression of genes whose products modulate transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. In other embodiments, the substances inhibit an activity of a product of such a gene. In some embodiments, the methods comprise adminstering a composition comprising a known inhibitor of calcineurin, and derivatives of these inhibitors that inhibit a phosphatase activity of calcineurn.